We performed confocal LSM using a commercial system (LSM510 Meta, Carl Zeiss). For readout of the sample, we used an oil-immersion objective (Plan-Apochromat 63×/1.40, Carl Zeiss). For this purpose, we put the immersion oil directly onto the sample. The microscope is equipped with two photomultiplier tubes, so that two color channels can be read out simultaneously. Thus, for samples emitting at four different fluorescence colors, each z-layer has to be read out twice using different filter sets. We chose the following alternating routine: For each z-layer, we first read out the blue and the orange channel using a chromatic beam splitter at λBS = 490 nm and two band-pass filters with 420 nm < λBP1 < 480 nm and 575 nm < λBP2 < 615 nm. Here, the sample was excited with a power of 236 μW at 405 nm and with a power of 109 μW at 488 nm. To scan the green and the red channel, we used a chromatic beam splitter at λ = 565 nm, a band-pass filter transparent between 505 nm < λBP3 < 550 nm for the green channel, and a long-pass filter with λLP > 650 nm for the red channel. For excitation, we used a blue laser (λ = 405 nm, P = 236 μW) and a red laser (λ = 633 nm, P = 28 μW). All excitation powers were measured in the back focal plane of the objective lens.

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